1. Field of the Invention
The present invention relates to materials and methods for the in vivo transport and delivery of nucleic acids. More particularly, it concerns the use of lipoproteins, including but not limited to, low density lipoproteins (xe2x80x9cLDLxe2x80x9d), and/or apolipoproteins for the in vivo transport of nucleic acids. In addition, the present invention relates to the use of lipoproteins in the early detection of cancer and/or metastatic cancer and/or arteriosclerosis.
2. Description of Related Art
The ultimate curative method for any genetic disorder, whether the disorder is inherited or results from a mutation, depends on an effective mode of replacing or augmenting non-functional gene(s). This process is now termed gene or genetic therapy. There are two important aspects to genetic therapy, the gene delivery system/vehicle and the gene control/expression program. Ideally, a replacement gene should become resident in the genome of the target cells/organism and be transferable to subsequent generations of cells and progeny, i.e., the change is incorporated into the germ cells or reproductive cells, the sperm and ovary. Although there have been several significant breakthroughs in this field, this area of biotechnology is still in its early development phase. The first step in any approach to gene replacement is the delivery of the specific gene (nucleic acid) to the cells.
Many techniques have been and are being developed to deliver and express genes in cells and specific tissues in mammals in vivo. Several general, non-specific methods for delivering genes have been reported involving aerosol nucleic acid delivery to cells (Stribling et al., 1992); calcium phosphate precipitation, using a steep change in ionic strength (Wigler et al., 1979); DEAE-dextran (Sompayrac et al., 1981); electroporation, forcing the nucleic acid into the cell by using an electric field or current (Neumann et al., 1982); microinjection, physically injecting the nucleic acid into a cell (Benvensty et al., 1986; Wolff et al., 1990); and polycationic molecules such as polylysine polypeptides (Curiel et al., 1992) and cationic lipids (Lee et al., 1996).
Liposomes, vesicles composed of synthetic or non-natural lipids such as long-chain fatty adds, can be used to carry the nucleic acid into the cell cytoplasm non-specifically (Felgner et al., 1987). A recent invention describes the delivery of a self-initiating and self-sustaining gene expression system which contains an RNA polymerase prebound to a DNA molecule using the aforementioned nucleotide delivery systems (U.S. Pat. No. 5,591,601).
Viral vectors in which specific nucleic acid sequences are incorporated into a neutralized or inactivated virus can use their viral entry mechanism to gain entry to the cell cytoplasm via specific cellular receptors to deliver nucleic acids (Schimotohono et al., 1981). The use of specific cellular receptors is apparently a more specific method for delivering genes. In this approach, the nucleic acid is bound either freely, through charge association, or alternatively it is chemically and non-reversibly conjugated to proteins with specific receptor proteins on the membrane of target cells for receptor-mediated uptake (Wu et al., 1988, Wu et al., 1989).
Techniques such as calcium phosphate precipitation, electroporation or DEAE-dextran transfection are not suitable for in vivo applications. Bombarding cells with nucleic acids under high pressure is a technique which has very limited applications in that it can only be applied topically and only a small number of cells can be targeted. Microinjection of nucleic acids into cells is mainly performed in vitro and requires actively dividing cells.
Gene delivery systems that use the viral entry mechanism of recombinant viral vectors have major disadvantages. Systems that utilize replication-defective adenoviral vectors can infect a wide variety of eukaryotic cell types including quiescent somatic cells utilizing the viral entry mechanism. However, adenoviral vector-based delivery systems are only successful in transient gene expression and repeated administration of the viral vector results in a strong immunological response of the host. In addition, the host will experience an adenoviral infection and can experience its symptoms if the recombinant vector undergoes homologous recombination with the wild-type virus strain. Systems that employ recombinant retroviral vectors can be used for stable integration of the gene of interest into the host""s genome, but only actively dividing cells can be targeted. In addition, the disadvantages of the adenoviral vector systems also apply to retroviral vector systems (immune response, disease etc.).
Positively-charged polycationic molecules such as polylysine peptides which bind non-specifically to the negatively charged nucleic acids have been used to introduce DNA into the chromosome of the recipient cell or organism. Cationic lipid vesicles, liposomes and micelles have been used in aggregates with DNA and viral envelope glycoproteins in non-specific, delivery of genes. Liposomes, vesicles composed of synthetic or non-natural lipids, such as long-chain fatty acids, can be used to carry the nucleic acid into the cell cytoplasm non-specifically. In these systems, the liposomes are structured to xe2x80x9cbest fitxe2x80x9d the nucleic acid and insertion into the cell is through non-specific uptake.
The interaction of the liposomal delivery systems discussed above with the nucleic acid to be delivered is non-specific. In addition, prior art techniques are designed to deliver multiple copies of the nucleic acid to the cell cytoplasm. Optimally, however, only one or two copies of a gene should be transfected per cell throughout the organism to replace a defective set of genes only in the specific cells and tissues where it would normally be expressed.
Thus there is a need for a safe and efficient gene delivery system that may be employed in the burgeoning filed of gene therapy.
The present invention contemplates a gene delivery system for use in gene therapy. Thus in particular embodiments, the present invention provides a composition comprising an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain; and a nucleic acid comprising an LDL or VLDL binding sequence, wherein the nucleic acid is bound to the polypeptide. In particularly preferred embodiments, the polypeptide comprises an LDL nucleic acid binding domain. In other embodiments, the polypeptide comprises a VLDL nucleic acid binding domain. In particular aspects of the present invention, the nucleic acid comprises an expression region operably linked to a promoter active in eukaryotic cells. In more particular embodiments, the expression region encodes a polypeptide. In other preferred embodiments, the expression region comprises an antisense construct.
In those embodiments in which the expression region encodes a polypeptide, the polypeptide may be selected from the group consisting of xcex1-globin, xcex2-globin, xcex3-globin, granulocytei macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, xcex2-interferon, xcex3-interferon, cytosine deaminase, adenosine deaminase, xcex2-glucuronidase, hypoxanthine guanine phosphoribosyl transferase, galactose-l-phosphate uridyltransferase, glucocerbrosidase, glucose-6-phosphatase, thymidine kinase, lysosomal glucosidase, growth hormone, nerve growth factor, insulin, adrenocorticotropic hormone, parathormone, follicle-stimulating hormone, luteinizing hormone, epidermal growth factor, thyroid stimulating hormone of CFTR, EGFR, VEGFR, IL-2 receptor, estrogen receptor, Bax, Bak, Bcl-Xs, Bik, Bid, Bad, Harakiri, Ad E1B, an ICE-CED3 protease neomycin resistance, luciferase, adenine phosphoribosyl transferase (APRT), retinoblastoma, insulin, mast cell growth factor, p53, p16, p21, MMAC1, p73, zac1 and BRCAI.
In those embodiments in which the expression region comprises an antisense construct, the antisense construct may be complementary to a segment of an oncogene. In more preferred embodiments, the oncogene may be selected from the group consisting of ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.
The expression region may be linked to a promoter selected from the group consisting of CMV IE, LTR, SV40 IE, HSV tk, xcex2-actin, human globin xcex1, human globin xcex2 and human globin y promoter. In a defined embodiment, the nucleic acid binding domain is an apoB100 nucleic acid binding domain. In other embodiments, the composition of the present invention may further comprise one or more lipoproteins selected from the group consisting of apoA1, apoA-II, apoA-IV, acat, apoE, apoC-II, apoC-III and apo-D. In particularly preferred embodiment, the apoB100 is selected from the group consisting of human, rat and baboon apoB 100.
In particular aspects of the invention, the polypeptide comprises at least two nucleic acid binding domains. In particularly preferred embodiments, the nucleic acid binding domain contains a motif selected from the group consisting of a proline pipe helix DNA binding motif, a ISGF3xcex3-like DNA binding motif, a SREBP-like DNA binding motif, a coiled-coil motif and a nucleotide (ATP)-binding motif. In more defined embodiments, the binding domain may be selected from the group consisting of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166 and SEQ ID NO:175.
In other embodiments, the polypeptide may further comprise at least one nuclear localization sequence. More particularly, the nuclear localization sequence may be from apoB100. In more preferred embodiments, the nuclear localization sequence may be selected from the group consisting of SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210.
Also contemplated by the present invention is a method for expressing a polypeptide in a human cell comprising the steps of providing a composition comprising (i) an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain and (ii) a nucleic acid comprising an expression cassette comprising a sequence encoding the polypeptide and a promoter active in eukaryotic cells, wherein the coding sequence is operably linked to the promoter, and wherein the nucleic acid sequence is bound to the LDL or VLDL; contacting the composition with the cell under conditions permitting transfer of the composition into the cell; and culturing the cell under conditions permitting the expression of the polypeptide.
In particularly preferred embodiments, the polypeptide independently, is a tumor suppressor, a cytokine, an enzyme, a hormone, a receptor, or an inducer of apoptosis. In preferred embodiments, the tumor suppressor may be selected from the group consisting of p53, p16, p21, MMAC1, p73, zac1, BRCAI and Rb. In preferred embodiments, the cytokine may be selected from the group consisting of IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-I5, TNF, GMCSF, xcex2-interferon and xcex3-interferon. In other preferred embodiments, the enzyme may be selected from the group consisting of cytosine deaminase, adenosine deaminase, xcex2-glucuronidase, hypoxanthine guanine phosphoribosyl transferase, galactose-1-phosphate uridyltransferase, glucocerbrosidase, glucose-6-phosphatase, thymidine kinase and lysosomal glucosidase. In still further preferred embodiments, the hormone may be selected from the group consisting of growth hormone, nerve growth factor, insulin, adrenocorticotropic hormone, parathormone, follicle-stimulating hormone, luteinizing hormone, epidermal growth factor and thyroid stimulating hormone. In defined embodiments, the receptor may be selected from the group consisting of CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor. In other preferred embodiments, the inducer of apoptosis may be selected from the group consisting of Bax, Bak, Bcl-Xs, Bik, Bid, Bad, Harakiri, Ad E1B and an ICE-CED3 protease.
In particularly preferred embodiments, the nucleic acid binding domain is an apoB 100 nucleic acid binding domain. In more preferred embodiments, the apoB 100 may be selected from the group consisting of human, rat and baboon low density apoB100. In still further preferred embodiments, the binding region is selected from the group consisting of a proline pipe helix DNA binding motif, a ISGF3xcex3-like DNA binding motif, a SREBP-like DNA binding motif, a coiled-coil motifs, and a nucleotide (ATP)-binding motif. In particular embodiments, the polypeptide further may comprise at least one nuclear localization sequence. In especially preferred embodiments, the nuclear localization sequence is derived from an apoB100 nuclear localization sequence. In specific embodiments, the polypeptide may be selected from the group consisting of xcex1-globin, xcex2-globin, xcex3-globin, neomycin resistance, luciferase, adenine phosphoribosyl transferase (APRT), and mast cell growth factor.
Also provided is a method for providing an expression construct to a human cell comprising providing a composition comprising (i) an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain and (ii) an expression cassette comprising a nucleic acid sequence encoding an expression region and a promoter active in eukaryotic cells, wherein the expression region is operably linked to the promoter, and wherein the nucleic acid sequence is bound to the LDL or VLDL; contacting the composition with the cell under conditions permitting transfer of the composition into the cell; and culturing the cell under conditions permitting the expression of the expression region.
In particularly preferred embodiments, the expression construct comprises an antisense construct. In more preferred embodiments, the antisense construct is derived from an oncogene. In exemplary embodiments, the oncogene may be selected from the group consisting ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl. In other embodiments, the expression construct comprises a nucleic acid coding for a gene. In preferred aspects the gene encodes a polypeptide.
In particularly preferred embodiments, the nucleic acid binding domain is an apoB100 nucleic acid binding domain. The apoB100 may be selected from the group consisting of human, rat and baboon low density apoB100. In other preferred embodiments, the DNA binding region is selected from the group consisting of a proline pipe helix DNA binding motif, a ISGF3xcex3-like DNA binding motif, a SREBP-like DNA binding motif, a coiled-coil motifs, and a nucleotide (ATP)-binding motif.
Further the present invention contemplates a method for treating a human disease comprising providing a composition comprising (i) an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain and (ii) an expression cassette comprising a nucleic acid sequence encoding an expression region and a promoter active in eukaryotic cells, wherein the expression region is operably linked to the promoter, and wherein the nucleic acid sequence is bound to the LDL or VLDL; and administering the composition to a human subject having the disease under conditions permitting transfer of the composition into cells of the human subject.
In specific embodiments, the disease may be selected from the group consisting of cancer, diabetes, cystic fibrosis and arteriosclerosis. In preferred embodiments the polypeptide comprises at least two nucleic acid binding regions. In other preferred embodiments the polypeptide comprises at least one nuclear localization sequence. In particularly preferred embodiments, the nucleic acid encodes a gene. In other preferred embodiments, the expression construct comprises an antisense construct.
Another aspects of the present invention describes a pharmaceutical composition comprising an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain; and a nucleic acid comprising an LDL or VLDL binding sequence, wherein the nucleic acid is bound to the polypeptide; the pharmaceutical composition being dispersed in a suitable diluent.
Also contemplated by the present invention is a method of transforming a cell comprising providing a cell; contacting the cell with a composition comprising (i) an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain and (ii) an expression cassette comprising a nucleic acid sequence encoding an expression region and a promoter active in eukaryotic cells, wherein the expression region is operably linked to the promoter, and wherein the nucleic acid sequence is bound to the LDL or VLDL; wherein expression of the expression region is indicative of the transformation.
Yet another aspect of the present invention contemplates a method of transfecting a cell comprising the steps of providing a cell; contacting the cell with a composition comprising (i) an isolated polypeptide comprising at least one LDL or VLDL nucleic acid binding domain and (ii) an expression cassette comprising a nucleic acid sequence encoding an expression region and a promoter active in eukaryotic cells, wherein the expression region is operably linked to the promoter, and wherein the nucleic acid sequence is bound to the LDL or VLDL; wherein expression of the expression region is indicative of the transfection.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.